The invention relates to assays used to confirm various ligands in a biological sample. The invention further relates to a microparticle neutralization assay which can be used to confirm ligands in a biological sample.
Several types of diagnostic assays for detecting or confirming ligands in a sample are currently available. An example of such assays includes direct sandwich immunoassays wherein a ligand-specific binding substance such as an antigen or antibody is coated on a solid phase and contacted with a biological sample thought to contain a ligand of interest. Next, the solid phase is contacted with a ligand-specific binding substance labeled with an appropriate label such as an enzyme, fluorescent label or radioisotope. The label can then be detected to determine the presence or quantity of ligand present in the sample. An example of a direct sandwich assay is Auszyme.RTM. immunoassay for detection of hepatitis B surface antigen available from Abbott Laboratories, Abbott Park, Ill.
Another example of an assay is a competitive or inhibition immunoassay wherein a ligand is detected in a biological sample by measuring the ligand's ability to compete with or inhibit binding of a ligand reagent for ligand-specific binding sites on a solid phase or labeled reagent. An example of a competitive immunoassay is Corab.RTM. immunoassay for antibody to hepatitis B core antigen also available from Abbott Laboratories.
Still another example of an assay for detecting ligands in a sample is the Western Blot procedure described by Towbin and Gordon, J. Immun. Meth., 72:313-340, 1984. This procedure involves electrophoresis of a known ligand-specific binding substance such as an antigen or antibody on sodium-dodecyl-sulfate polyacrylamide gels (SDS-PAGE). The ligand-specific binding molecule generates a characteristic banding pattern on the gel. This banding pattern is transferred under an electric current from the SDS-PAGE to nitrocellulose filter paper. The filter paper is then incubated with a biological sample containing a ligand of interest. Any ligand in the sample specific for the known ligand-specific binding substance binds to the nitrocellulose filter paper to form a complex such as an antigen-antibody complex. The antigen-antibody complex with its characteristic banding pattern is visualized using a labeled ligand-specific binding substance against the antigen-antibody complex. The Western Blot procedure is a very time-consuming and technique-sensitive procedure involving expensive equipment and highly trained personnel. Therefore, this procedure is not feasible for many laboratories.
Yet another type of assay is a neutralization procedure for confirming samples thought to be positive for a ligand of interest. A ligand-specific binding substance, usually an antibody or antigen, is used as a neutralizing reagent and is added to the biological sample which has previously been tested as positive by another assay method. In a truly positive sample, the neutralization reagent binds to the ligand of interest and prevents it from reacting with any other reagents. The biological sample containing the neutralization reagent is then assayed in an immunoassay such as those described above. A reduction in the ligand previously detected indicates the sample was neutralized and is a true positive. This neutralization procedure works well for confirming certain assays, for example, hepatitis B surface antigen assays such as Auszyme.RTM. II confirmatory neutralization assay, Abbott Laboratories. However, one problem with such assays is that the neutralization reagent can react nonspecifically with the solid phase of the immunoassay yielding equivocal results.